The Yariv reagent: behaviour in different solvents and interaction with a gum arabic arabinogalactan-protein.

The ß-D-Glc Yariv reagent is frequently used to isolate and to study the structure of arabinogalactan-proteins with the arabinogalactan type II structure. The present paper describes the aggregation features of the Yariv reagent in water, salt solutions and in organic solvents as determined by NMR, absorption spectroscopy and light scattering experiments. The results indicate that in water the Yariv reagent forms aggregates of up to 300 units and in 1% aqueous NaCl the degree of aggregation is approx. 150. The aggregates are formed both by H-bonds and hydrophobic interactions, the former appearing to be of most importance in water. The interaction between the Yariv reagent and an AGP fraction from gum arabic, showed a degree of aggregation of the Yariv reagent when using 1% NaCl to be of approx. 150 units, whereas disruption of the aggregate took place in 10% NaCl with an aggregation number of approx. 100. Partial acid hydrolysis of an AGP from gum Arabic (Acacia Senegal) and analyses of the linkage types remaining indicated that a certain length of (1→3)-ß-linked galactose units was necessary for binding between the Yariv reagent and the AGP. This is in accordance to what also was recently observed by Kitazawa et al. (2013).

Curdlan and other bacterial (1-->3)-beta-D-glucans.

Three structural classes of (1-->3)-beta-D-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (1-->3)-beta-glucans and side-chain-branched (1-->3,1-->2)-beta-glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (1-->3,1-->6)-beta-glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (1-->3)-beta-glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (1-->3)-beta-glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.

Structure of epiglucan, a highly side-chain/branched (1 --> 3;1 --> 6)-beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht.

The extracellular fungal polysaccharide, epiglucan, synthesised by Epicoccum nigrum is a side-chain/branched (1 --> 3;1 --> 6)-D-beta-glucan. Methylation analysis, 13C DEPT NMR and specific enzymic digestion data show slight variation in branching frequency among the epiglucans from the three strains examined. The (1 --> 3)-beta-linked backbone has (1 --> 6)-beta-linked branches at frequencies greater than the homologous glucans, scleroglucan and schizophyllan, from Sclerotium spp. and Schizophyllum commune, respectively. The structural analyses do not allow a distinction to be made between structures I and II. [structures: see text] Epiglucan displays non-Newtonian shear thinning rheological properties, typical of these glucans.

A (1-->4)-beta-mannan-specific monoclonal antibody and its use in the immunocytochemical location of galactomannans.

Galactomannan was coupled to a protein carrier for the preparation of monoclonal antibodies. The monoclonal antibodies generated bound to galactomannans from different sources as well as to glucomannan and galactoglucomannan. One monoclonal antibody, BGM C6, was characterised and found to be specific for (1-->4)-beta-linked mannopyranosyl residues; it had a binding affinity estimated at 1x10(-6) M for the (1-->4)-beta-linked mannohexaose. BGM C6 was used in immunogold labelling studies to locate galactomannans in the endosperm walls of normal coconuts (Cocos nucifera L.) and those of the mutant makapuno at two different developmental stages. The pattern and intensity of antibody labelling varied for each type of coconut at the mature and immature stages, indicating differences in the galactomannan composition of the endosperm walls.

Determinants of the outcome of intrauterine insemination: analysis of outcomes of 9963 consecutive cycles.

OBJECTIVE: Our aim was to determine which factors influence the effectiveness of intrauterine insemination. STUDY DESIGN: This article is a retrospective statistical analysis of outcomes of 9963 consecutive intrauterine insemination cycles. RESULTS: Patient age was the main determinant of pregnancy outcome (analysis of variance F ratio = 29, P <.0001), followed by the number of follicles at the time of intrauterine insemination (analysis of variance F ratio = 9, P <.0001) and sperm motility in the inseminate (analysis of variance F ratio = 4, P =.002). A total of 18.9% of all patients <26 years old conceived, compared with 13.9% of those 26-30 years old, 12.4% of those 31-35 years old, 11.1% of those 36-40 years old, 4.7% of those 41-45 years old, and 0.5% of patients >45 years old (P <.001). When analyzed by single years, ongoing pregnancy rates after intrauterine insemination remained high through age 32 years. Across all ages and causes of infertility, 7.6% of patients with 1 follicle at the time of intrauterine insemination conceived, compared with 10. 1% with 2, 14.0% with 4, and 16.9% with 6 follicles (P <.01). When ovulation occurred before intrauterine insemination (ie, no visible follicular structures), 4.6% of patients conceived. The likelihood of pregnancy was maximized when motile sperm numbers were >/=4 million and sperm motility was >/=60%. Differences in pregnancy outcomes between sperm processing options were related to differences in sperm motility after processing; use of methods incorporating motility enhancement with pentoxifylline and motile sperm concentration through silica gradients yielded the highest overall pregnancy rates. CONCLUSION: When the results of ongoing retrospective analysis of intrauterine insemination outcomes are applied, overall intrauterine insemination pregnancy rates have increased from 5.8% per cycle in 1991 to 13.4% per cycle in 1996, during which time the average age of patients undergoing intrauterine insemination has increased from 36.1 (+/-0.2) to 39.2 (+/-0.1) years.

The influence of gender on incidence and outcome of patients with bladder cancer in Harlem.

Although African Americans have a lower incidence of bladder cancer, overall survival is worse compared with American whites. This phenomenon has been attributed to the higher incidence of advanced disease at diagnosis and poor follow-up. Fifty-nine cases of bladder cancer were identified through the Tumor Registry at Harlem Hospital and reviewed retrospectively. Complete data were obtained for 42 patients. The primary independent variables of interest were primary care utilization, comorbid conditions, social variables, and gender. The outcome variables of interest were stage of disease at presentation and death. The median age at diagnosis in this group was 73 years compared with 68 for bladder cancer patients in the United States. There was no statistically significant correlation between primary care utilization or severity of comorbidities, and clinical stage at presentation. Similarly, these variables did not influence the occurrence of death as an outcome. For women, the mean age at diagnosis was 74.2 years compared with 67.3 in men (P = .112). The ratio of male-to-female cases in this group was 1.3 to 1 compared with 2.7 to 1 for the general US population. Women had lower odds of being diagnosed with superficial disease (OR = 0.24, 95% CI, 0.06-0.94) and a higher incidence of a cancer-specific death (OR = 2.7, 95% CI). The poor outcome and high incidence of bladder cancer cases among women in Harlem is intriguing. Overall, primary care utilization, comorbidities, and other social factors did not seem to influence stage or death as an outcome. The significantly elevated prevalence of smoking among women in this community, increased age at diagnosis, and possible environmental influences may play a role.

Field stimulation of isolated chick heart cells: comparison of experimental and theoretical activation thresholds.

INTRODUCTION: This study examines the accuracy of using membrane models to predict activation thresholds for chick heart cells during field stimulation. METHODS AND RESULTS: Activation thresholds were measured experimentally in ten embryonic chick heart cells at 37 degrees C for stimulus durations 0.2 to 40 msec. Activation was assessed by observing the mechanical twitch of the cell. The heart cells ranged in diameter from 15.0 to 26.7 microm. Since the electric field required for activation depends on diameter, the thresholds were expressed as the maximum field-induced transmembrane potential, Vth = 1.5 a Eth, where a is the cell radius and Eth is the strength of the electric field at threshold. A cell model was created using a singular perturbation method and membrane models describing the ionic currents of a heart cell. The study used membrane models of Ebihara and Johnson (1980), Luo and Rudy (1991), Shrier and Clay (1994), and their combinations. The results show that for stimuli longer than 1 msec, theoretical activation thresholds were within one standard deviation of experimental thresholds. For shorter stimuli, the models failed to predict thresholds because of a premature deactivation of the sodium current. The modification of the m gates dynamics, so that they closed with a time constant of 1.4 msec, allowed to predict thresholds for all durations. The root mean square error between experimental and theoretical thresholds was 6.14%. CONCLUSIONS: The existing membrane models can predict thresholds for field stimulation only for stimuli longer than 1 msec. For shorter stimuli, the models need a more accurate representation of the sodium tail current.

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene.

Genes essential for the production of a linear, bacterial (1-->3)-beta-glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)-beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.

Cell wall (1-->3)- and (1-->3, 1-->4)-beta-glucans during early grain development in rice (Oryza sativa L.).

Immunogold labeling was used to study the distribution of (1-->3)-beta-glucans and (1-->3, 1-->4)-beta-glucans in the rice grain during cellularization of the endosperm. At approximately 3-5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1-->3)-beta-glucans but not (1-->3, 1-->4)-beta-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1-->3, 1-->4)-beta-glucans with (1-->3)-beta-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state.

Specificity of binding of beta-glucoside activators of ryegrass (1-->3)-beta-glucan synthase and the synthesis of some potential photoaffinity activators.

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1-->3)-beta-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a beta-D-glycosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in beta-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single beta-D-glucosyl residue, and beta-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charge aglycons increase Ka. Relative to methyl-beta-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-beta-D-glucosylamine and 2'-[4-azidosalicylamino]ethyl-1-thio-beta-D-glucoside with K(a)s of approximately 30 microM. The latter also was tested for its potential to specifically label the beta-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.

Purification and characterization of an extracellular (1 --> 6)-beta-glucanase from the filamentous fungus Acremonium persicinum.

An endo-(1 --> 6)-beta-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 --> 6)-beta-glucans (pustulan and lutean), initially yielding a series of (1 --> 6)-beta-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as beta-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent bydrolysis of (1 --> 6)-beta- and some (1 --> 3)-beta-linkages in this substrate. K(m) values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

Pharmaceutical services for a homeless population.

Participation of pharmacist volunteers in the medication program of a countrywide health care program for homeless persons is described. Pharmacist volunteers were brought in to manage medications for a health care program serving homeless persons in Ramsey County, Minnesota. After the pharmacy program was structured, volunteers were recruited from the community. Pharmacists duties initially focused on product management but were expanded to include establishing and monitoring the program formulary; reviewing patient records and prescriptions for allergies, potential drug interactions, and appropriate dosage; counseling patients on medication use; and consulting with other members of the health care team. The pharmacists' efforts led to improvements in monitoring and stocking of necessary medications. The cost of the pharmacy program decreased from $1800 a month to as little as $300 a month. The value of donated supplies and medications increased from $8,600 in 1991 to over $122,000 in 1994. Pharmacist volunteers helped to improve the cost-effectiveness and quality of medication use in a homeless population.

Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries.

Antisense oligonucleotides can vary significantly and unpredictably in their ability to inhibit protein synthesis. Libraries of chimeric oligonucleotides and RNase H were used to cleave and thereby locate sites on human multidrug resistance-1 RNA transcripts that are relatively accessible to oligonucleotide hybridization. In cell culture, antisense sequences designed to target these sites were significantly more active than oligonucleotides selected at random. This methodology should be generally useful for identification of potent antisense sequences. Correlation between oligonucleotide activity in the cell culture assay and in an in vitro RNase H assay supports the proposed role of the enzyme in the mechanism of antisense suppression in the cell.

Barley beta-D-glucan exohydrolases with beta-D-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone.

Two beta-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH2-terminal amino acids show 64% positional identity. Both enzymes hydrolyze the (1,3)-beta-glucan, laminarin, but also hydrolyze (1,3;1,4)-beta-glucan and 4-nitrophenyl beta-D-glucoside. The complete sequence of 602 amino acid residues of the mature beta-glucan exohydrolase isoenzyme ExoII has been deduced by nucleotide sequence analysis of a near full-length cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated betaI and betaII, were also purified from the extracts. Their amino acid sequences are similar to enzymes classified as beta-glucosidases and although they hydrolyze 4-nitrophenyl beta-glucoside, their substrate specificities and action patterns are more typical of polysaccharide exohydrolases of the (1,4)-beta-glucan glucohydrolase type. Both the beta-glucan exohydrolase isoenzyme ExoI and the beta-glucosidase isoenzyme betaII release single glucosyl residues from the nonreducing ends of substrates and proton-NMR shows that anomeric configurations are retained during hydrolysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and glucosidases, together with more specific questions regarding the functional roles of the two classes of enzyme in germinating barley grain.